| 1. |
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| 2. |
- Gunnarsson, Anders, 1981, et al.
(författare)
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Time-resolved surface-enhanced ellipsometric contrast imaging for label-free analysis of biomolecular recognition reactions on glycolipid domains
- 2012
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 84:15, s. 6538-6545
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Tidskriftsartikel (refereegranskat)abstract
- We have applied surface-enhanced ellipsometry contrast (SEEC) imaging for time-resolved label-free visualization of biomolecular recognition events on spatially heterogeneous supported lipid bilayers (SLB). Using a conventional inverted microscope equipped with total internal reflection (TIR) illumination, biomolecular binding events were monitored with a lateral resolution near the optical diffraction limit at an acquisition rate of ∼1 Hz with a sensitivity in terms of surface coverage of ∼1 ng/cm2. Despite the significant improvement in spatial resolution compared to alternative label-free surface-based imaging technologies, the sensitivity remains competitive with surface plasmon resonance (SPR) imaging and imaging ellipsometry. The potential of the technique to discriminate local differences in protein binding kinetics was demonstrated by time-resolved imaging of anti-GalCer antibodies binding to phase-separated lipid bilayers consisting of phosphatidylcholine (POPC) and galactosylceramide (GalCer). A higher antibody binding capacity was observed on the GalCer-diluted fluid region in comparison to the GalCer-rich gel phase domains. This observation is tentatively attributed to differences in the presentation of the GalCer epitope in the two phases, resulting in differences in availability of the ligand for antibody binding. The complementary information obtained by swiftly switching between SEEC and fluorescence (including TIR fluorescence) imaging modes was used to support the data interpretation. The simplicity and generic applicability of the concept is discussed in terms of microfluidic applications.
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| 3. |
- Feuz, Laurent, 1975, et al.
(författare)
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Improving the Limit of Detection of Nanoscale Sensors by Directed Binding to High-Sensitivity Areas
- 2010
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Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 4:4, s. 2167-2177
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Tidskriftsartikel (refereegranskat)abstract
- The revelation of protein protein-interactions is one of the main preoccupations in the field of proteomics. Nanoplasmonics has emerged as an attractive surface-based technique because of its ability to sense protein binding under physiological conditions in a label-free manner. Here, we use short-range ordered holes with a diameter of similar to 150 nm and a depth of similar to 50 nm as a nanoplasmonic template. A similar to 40 nm high cylindrical region of Au is exposed on the walls of the holes only, while the rest of the surface consists of TiO2. Since the sensitivity is confined to the nanometric holes, the use of two different materials for the sensor substrate offers the opportunity to selectively bind proteins to the most sensitive Au regions on the sensor surface. This was realized by applying material-selective poly(ethylene glycol)-based surface chemistry, restricting NeutrAvidin binding to surface-immobilized biotin on the Au areas only. We show that under mass-transport limited conditions (low nM bulk concentrations), the initial time-resolved response of uptake could be increased by a factor of almost 20 compared with the case where proteins were allowed to bind on the entire sensor surface and stress the generic relevance of this concept for nanoscale sensors. In the scope of further optimizing the limit of detection (LOD) of the sensor structure, we present finite-element (FE) simulations to unravel spatially resolved binding rates. These revealed that the binding rates in the holes occur in a highly inhomogeneous manner with highest binding rates observed at the upper rim of the holes and the lowest rates observed at the bottom of the holes. By assuming a plasmonic field distribution with enhanced sensitivity at the Au-TiO(2)interface, the FE simulations reproduced the experimental findings qualitatively.
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| 4. |
- Gunnarsson, Anders, 1981, et al.
(författare)
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Kinetics of Ligand Binding to Membrane Receptors from Equilibrium Fluctuation Analysis of Single Binding Events
- 2011
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 133:38, s. 14852-14855
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Tidskriftsartikel (refereegranskat)abstract
- Equilibrium fluctuation analysis of single binding events has been used to extract binding kinetics of ligand interactions with cell-membrane bound receptors. Time-dependent total internal reflection fluorescence (TIRF) imaging was used to extract residence-time statistics of fluorescently stained liposomes derived directly from cell membranes upon their binding to surface-immobilized antibody fragments. The dissociation rate constants for two pharmaceutical relevant antibodies directed against different B-cell expressed membrane proteins was clearly discriminated, and the affinity of the interaction could be determined by inhibiting the interaction with increasing concentrations of soluble antibodies. The single-molecule sensitivity made the analysis possible without overexpressed membrane proteins, which makes the assay attractive in early drug-screening applications.
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| 5. |
- Jönsson, Peter, 1981, et al.
(författare)
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Accumulation and Separation of Membrane-Bound Proteins Using Hydrodynamic Forces
- 2011
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 83:2, s. 604-611
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Tidskriftsartikel (refereegranskat)abstract
- The separation of molecules residing in the cell membrane remains a largely unsolved problem in the fields of bioscience and biotechnology. We demonstrate how hydrodynamic forces can be used to both accumulate and separate membrane-bound proteins in their native state. A supported lipid bilayer (SLB) was formed inside a microfluidic channel with the two proteins streptavidin (SA) and cholera toxin (CT) coupled to receptors in the lipid bilayer. The anchored proteins were first driven toward the edge of the lipid bilayer by hydrodynamic forces from a flowing liquid above the SLB, resulting in the accumulation of protein molecules at the edge of the bilayer. After the concentration process, the bulk flow of liquid in the channel was reversed and the accumulated proteins were driven away from the edge of the bilayer. Each type of protein was found to move at a characteristic drift velocity, determined by the frictional coupling between the protein and the lipid bilayer, as well as the size and shape of the protein molecule. Despite having a similar molecular weight, SA and CT could be separated into monomolecular populations using this approach. The method also revealed heterogeneity among the CT-molecules, resulting in three subpopulations with different drift velocities. This was tentatively attributed to multivalent interactions between the protein and the monosialoganglioside G(M1) receptors in the lipid bilayer.
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| 6. |
- Jönsson, Peter, 1981, et al.
(författare)
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Sealing of Submicrometer Wells by a Shear-Driven Lipid Bilayer
- 2010
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 10:5, s. 1900-1906
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Tidskriftsartikel (refereegranskat)abstract
- A supported lipid bilayer (SLB) was formed in a microfluidic channel by vesicle fusion. The SLB, formed on a flat part of the surface, was driven by the shear forces of a bulk flow above the SLB to a part of the surface with embedded submicrometer wells. When using a bulk solution with a pH of 9.5 the advancing lipid bilayer sealed the wells, creating free-spanning membranes, whereas at a pH of 8.0 the SLB instead followed the contour of the wells.
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| 7. |
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| 8. |
- Simonsson, Lisa, 1982, et al.
(författare)
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Continuous Lipid Bilayers Derived from Cell Membranes for Spatial Molecular Manipulation
- 2011
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 133:35, s. 14027-14032
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Tidskriftsartikel (refereegranskat)abstract
- Progress with respect to enrichment and separation of native membrane components in complex lipid environments, such as native cell membranes, has so far been very limited. The reason for the slow progress can be related to the lack of efficient means to generate continuous and laterally fluid supported lipid bilayers (SLBs) made from real cell membranes. We show in this work how the edge of a hydrodynamically driven SLB can be used to induce rupture of adsorbed lipid vesicles of compositions that typically prevent spontaneous SLB formation, such as vesicles made of complex lipid compositions, containing high cholesterol content or being derived from real cell membranes. In particular, upon fusion between the moving edge of a preformed SLB and adsorbed vesicles made directly from 3T3 fibroblast cell membranes, the membrane content of the vesicles was shown to be efficiently transferred to the SLB. The molecular transfer was verified using cholera toxin B subunit (CTB) binding to monosialoganglioside receptors (G(M1) and G(M3)), and the preserved lateral mobility was confirmed by spatial manipulation of the G(M1/M3)-CTB complex using a hydrodynamic flow. Two populations of CTB with markedly different drift velocity could be identified, which from dissociation kinetics data were attributed to CTB bound with different numbers of ganglioside anchors.
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| 9. |
- Johansson Fast, Björn, 1986, et al.
(författare)
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Hydrodynamic separation of proteins in supported lipid bilayers confined by gold barriers
- 2013
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Ingår i: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 9:39, s. 9414-9419
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Tidskriftsartikel (refereegranskat)abstract
- Hydrodynamic drag forces generated by liquid flow above a supported lipid bilayer (SLB) can be used to induce lateral movement of molecules protruding from the SLB. Since the velocity of the individual molecules depends on their size and coupling to the lipid bilayer, these forces can also be used to enrich and separate different types of membrane-bound molecules. To improve and better quantify hydrodynamic-based molecular separation in SLBs, we formed the SLB on the floor of a microfluidic channel which was patterned with gold barriers that confined the lipid bilayer to a 100 mm wide strip in the center of a 300 mm wide microfluidic channel. This forces the SLB into a region of the channel where the spatial variation of the hydrodynamic forces is close to zero while at the same time preventing the SLB from creeping up on the PDMS sides of the channel, thus reducing the loss of material. We here use this approach to investigate the accumulation of (i) fluorescently labeled lipids and (ii) the protein complex cholera toxin B (CTB) and to compare how the accumulation and separation differ when having an infinite reservoir or only a spatially limited band of studied molecules in the SLB. In addition, we show how the method can be used for complete separation of different polyvalently bound fractions of CTB.
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| 10. |
- Johansson Fast, Björn, 1986, et al.
(författare)
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Label-Free Measurements of the Diffusivity of Molecules in Lipid Membranes
- 2014
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Ingår i: ChemPhysChem. - : Wiley. - 1439-7641 .- 1439-4235. ; 15:3, s. 486-491
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Tidskriftsartikel (refereegranskat)abstract
- An important and characteristic property of a cell membrane is the lateral mobility of protein molecules in the lipid bilayer. This has conventionally been measured by labeling the molecules with fluorescent markers and monitoring their mobility by different fluorescence-based techniques. However, adding the label to the studied molecule may affect the system, so it is an assumption in almost all experiments that the measured mobility of the biomolecule with its label is the same as that of the unlabeled molecule. However, this assumption is rarely tested due to a lack of suitable methods. In this work, a new technique to perform label-free diffusivity measurements is developed and used to measure the effect of the label for two common protein-lipid systems: 1) streptavidin (SA) coupled to a supported lipid bilayer (SLB) through biotinylated lipids and 2) the extracellular part of the T-cell adhesion protein CD2, coupled to an SLB through histidine tags to nickel-chelating lipids. A measurable (approximate to 12%) decrease in diffusivity is found for both labeled proteins, even though the molecular mass of the label is almost 100 times smaller than those of the proteins (approximate to 50 kDa). The results illustrate the importance of being able to study different biophysical properties of cell membranes and their mimics without relying on fluorescent labels, especially if fluorescent labeling is difficult or is expected to affect the nature of the intermolecular interactions being studied.
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